Methods for preventing graft rejection in transplantation and for producing a universal gene therapy host cell using lymphocyte activation (lag-3)

ABSTRACT

A method to prevent graft rejection of transplanted cells, tissues or organs without general immunosuppression is described. The method employs a newly discovered protein, LAG-3. When allogeneic or xenogeneic cells are engineered to express LAG-3 on their surface and transplanted, immune destruction of the implanted cell, tissue or organ is prevented, while the host&#39;s immune system remains functional. A particular application of this method allows the preparation of a universal gene therapy host cell expressing LAG-3 on its surface for protection from graft rejection by a host&#39;s immune system.

FIELD OF THE INVENTION

[0001] The present invention relates to methods for preventing graftrejection of transplantated organs, tissues or cells, in particular tosuch methods which comprise engineering a cell type to express a LAG-3protein when transplanted in a host. More particularly, the inventionrelates to the production of a universal gene therapy host cellexpressing the LAG-3 protein on its surface.

DESCRIPTION OF THE BACKGROUND ART

[0002] The lymphocyte activation gene (LAG-3) is a member of theimmunoglobulin superfamily, that is selectively transcribed in humanactivated T (both CD4⁺ and CD8⁺) and NK cells (Triebel et al, 1990 ).The sequence data, the compared exon/intron organization, and thechromosomal localization revealed that LAG-3 is closely related to CD4(Baixeras et al, 1992). The close relationship between LAG-3 and CD4 wasfurther strengthened by the demonstration that both share the sameligand, i.e., MHC class II molecules (Baixeras et al, 1992). However, incontrast to CD4, LAG-3 does not bind the human immunodeficiency virusgp120 (Baixeras et al, 1992). In vivo, LAG-3 expression was neitherfound in primary lymphoid organs, such as spleen, mucosa-associatedlymphoid tissue or normal lymph nodes. However, it was readily detectedin inflamed tonsils, or lymph nodes with follicular hyperplasia,supporting the view that even in vivo LAG-3 is expressed followingactivation (Huard et al, 1994A). Antigen-specific stimulation of T-cellclones in the presence of anti-LAG-3 monoclonal antibody (mAb) led toincreased thymidine incorporation, higher expression of activationmarker CD25 and enhanced cytokine production (Huard et al, 1994B).

[0003] Accordingly, addition of a soluble recombinant form of LAG-3inhibited antigen-specific T-cell proliferation, suggesting a regulatoryrole of LAG-3 in CD4⁺ T-lymphocyte activation (Huard, 1996) and itsinvolvement in extinguishing ongoing immune responses. Recently, it hasbeen shown that LAG-3 also acts as a co-receptor for NK cells anddefines different modes of tumor cell killing controlled by the innateimmune system (Miyazaki et al., 1996).

[0004] The mechanics by which a T cell response to a foreign (allogeneicor xenogeneic) protein or cell or organ is mounted are fairly wellunderstood. Antigen presenting cells (APCs) are attracted to areas ofinflammation or damage (that may be induced by surgicaltransplantation). The repertoire of T cells in the periphery isconstantly surveying tissues for evidence of pathogens or the presenceof foreign (allo- or xenogeneic) tissue. Once any of these warningsignals are recognized, the APCs engulf the protein, digest it andpresent it to the host's immune system.

[0005] Allogeneic or syngenic tumor cells have been engineered toexpress viral IL-10 which induces local anergy to the tumors. Such atreatment did not affect the rejection of a non-transduced tumor at adistant site ( Suzuki et al., 1995). IL-10 delivered locally is thoughtto shift the T cell repertoire reactive to the transplanted cells to aTh2 phenotype that is not cytolytic and may even be protective.

[0006] Cells naturally expressing the Fas ligand have been transplantedacross allogeneic or exogeneic barriers without immunosuppression.Surveillance of the site of implantation by host T cells results intheir killing when contacted by Fas ligand (Bellgrau et al., 1995).Moreover, rejection of pancreatic islet allografts has been prevented bythe cotransplantation of syngeneic myoblasts genetically engineered toexpress the Fas ligand (Lau et al., 1996)

[0007] The immune system is well equipped to rapidly identify foreign,diseased or inflamed tissue and rapidly destroys it. This has alwaysbeen a major barrier to tissue, organ and cell transplantation as wellas gene therapy. Major problems are generally associated with chronicimmunosuppression, encapsulation or immunoisolation. The unwanted sideeffects of chronic immunosuppression include increased susceptibility toopportunistic infection and tumor formation.

[0008] The desire for long-term acceptance of grafted tissue in theabsence of continuous immunosuppression is a long-standing goal in humanmedicine.

[0009] Citation of any document herein is not intended as an admissionthat such document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

SUMMARY OF THE INVENTION

[0010] It has now been found that transplantation of cells that expressa LAG-3 protein on their surface results in the protection from graftrejection by the host's immune system.

[0011] The present invention thus provides a genetically engineered cellwhich may be part of a tissue or organ to be transplanted, comprisingDNA encoding a transmembrane LAG-3 protein on its surface, resulting inthe protection from graft rejection by the host's immune system, the DNAbeing genomic DNA or cDNA. Said DNA can be exogenous or, in a particularembodiment of the invention, the endogenous DNA, whose expression isactivated or modified through the targeted insertion of a regulatorysequence and/or an amplifiable gene by homologous recombination. TheLAG-3 protein is a protein which is recognized by antibodies directedagainst LAG-3.

[0012] When the cell is part of the tissue or organ to be transplanted,transfection of the LAG-3 DNA can be accomplished directly on the tissueor organ to be transplanted.

[0013] In particular, the cell is a universal gene therapy host cell,suitable, for example, for any kind of somatic or “ex vivo” genetherapy.

[0014] In a specific embodiment, the gene therapy host cell furthercomprises exogenous DNA encoding a therapeutic agent of interest, andthe genetically engineered cells are employed as a therapeutic agent.The term “therapeutic” as used herein, includes treatment and/orprophylaxis.

[0015] In a further embodiment, the gene encoding a therapeutic agent ofinterest is present in the genome of the cell and the cell furthercomprises exogenous DNA encoding a regulatory sequence or an amplifiablegene for activating or modifying the expression of the endogenous geneof interest.

[0016] The genetically engineered cell of the invention can, anyway,contain the exogenous LAG-3 DNA only, to be used in a mixture with othergene therapy host cells containing the therapeutic DNA of interest.

[0017] The cell of the present invention is preferably selected frommyoblasts, fibroblasts, hematopoietic stem cells, embryonic stem cells,foetal liver cells, umbilical vein endothelial cells and CHO cells.

[0018] Cells as above, deriving from transgenic animals, are also withinthe scope of the present invention.

[0019] It is a further object of the present invention the use of atransmembrane LAG-3 protein, including muteins and variants thereof,expressed on the surface of the cells, in the manufacture of amedicament to induce protection from graft rejection by a host's immunesystem.

[0020] Furthermore, the present invention provides the use of a cellcomprising DNA encoding a transmembrane LAG-3 protein, expressed on thesurface of the cell, in the manufacture of a medicament to induceprotection from graft rejection by a host's immune system.

[0021] The use of said cell expressing LAG-3 on its surface, in themanufacture of a medicament to be mixed with cells, tissues or organs tobe transplanted, to induce protection from graft rejection by a host'simmune system, is also within the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1—Cytotoxic activity versus LH-CHO cells of splenocytes frommice primed with LAG-3-CHO or LH-CHO cells. The mean value (±SD) from 5mice primed as indicated in the legend; 2 non-primed mice were alsoevaluated.

[0023]FIG. 2±Cytotoxic activity versus LAG-3-CHO cells of splenocytesfrom mice primed with LAG-3-CHO or LH-CHO cells. The mean value (±SD)from 5 mice primed as indicated in the legend; 2 non-primed mice werealso evaluated.

[0024]FIG. 3—Map of the Dα mammalian expression vector. Abbreviationused: DHFR, dehydrofolate transcription unit (Subraimani et al., 1981);pML, derivative of pBR322 (Lusky and Botchan, 1981); hAIVSA, fragmentfrom intron A of the α subunit of human glycoprotein hormones (Fiddesand Goodman, 1981); MMT-1, promoter of the mouse metallothionein 1(Hamer and Walling, 1982).

DETAILED DESCRIPTION OF THE INVENTION

[0025] Hundreds of thousands of people die each year as a result of aheart, kidney, liver, lung or pancreas failure. The single mosteffective therapy is transplantation.

[0026] Therapy associated to transplantation of cells, tissues or organsinduces a general immuno-protective state in the host relative to theengrafted cell, tissue or organ. It is desirable to establish graftspecific protection against rejection by the host's immune systemparticularly in allogeneic transplantation, xenogeneic transplantationand gene therapy. Also, it is desirable to inhibit tolerance to tumortissue or otherwise allow the host's immune system to attack tumortissue.

[0027] Accordingly, the present invention is directed to all of themethods described above for using the finding that transplantation ofcells or tissues which express a transmembrane LAG-3 protein results inthe protection of graft rejection by the host's immune system.

[0028] The invention employs the newly discovered gene and protein,LAG-3, that is normally expressed on activated T cells and activated NKcells.

[0029] The definition “transmembrane LAG-3 protein” as used hereinrefers to any transmembrane protein containing the extracytoplasmaticdomain of LAG-3, its salts, functional derivatives, precursors andactive fractions as well as its active mutants and its active variants,which are all expressed on the surface of a cell.

[0030] The definition also refers to a transmembrane protein expressedin its natural state or can be fused, for example by geneticengineering, to another protein, such as a glycosyl phosphatidylinositolanchor or any relevant fragments of another transmembrane protein, forexample TNF-receptor, MPL-ligand or a transmembrane immunoglobulin.

[0031] The definition “salts” as used herein refers both to salts of thecarboxyl-groups and to salts of the amino functions of the compoundobtainable through known methods. The salts of the carboxyl-groupscomprise inorganic salts as, for example, sodium, potassium, calciumsalts and salts with organic bases as those formed with an amine astriethanolamine, arginine or lysine. The salts of the amino groupscomprise, for example, salts with inorganic acids as hydrochloric acidand with organic acids as acetic acid.

[0032] The definition “functional derivatives” as herein used refers toderivatives which can be prepared from the functional groups present onthe lateral chains of the amino acid moieties or on the terminal N- orC- groups according to known methods and are comprised in the inventionwhen they are pharmaceutically acceptable, i.e. when they do not destroythe protein activity or do not impart toxicity to the pharmaceuticalcompositions containing them. Such derivatives include, for example,esters or aliphatic amides of the carboxyl-groups and N-acyl derivativesof free amino groups or 0-acyl derivatives of free hydroxyl-groups andare formed with acyl-groups as, for example, alcanoyl- or aroyl-groups.

[0033] The “precursors” are compounds which are converted into LAG-3 inthe human or animal body.

[0034] As “active fractions” of the protein the present invention refersto any fragment or precursor of the polypeptidic chain of the compounditself, alone or in combination with related molecules or residues boundto it, for example residues of sugars or phosphates, or aggregates ofthe polypeptide molecule when such fragments or precursors show the sameactivity of LAG-3 as medicament.

[0035] Preferred “active fractions” are soluble fractions from theextracellular portion of the LAG-3 protein, including one or more of thefour domains, D1, D2, D3, D4, of the extracytoplasmatic domain of LAG-3.

[0036] The definition “active mutants” as used herein refers to otherproteins or polypeptides wherein one or more amino acids of thestructure are eliminated or substituted by other amino acids, or one ormore amino acids are added to that sequence in order to obtainpolypeptides or proteins having the same activity of LAG-3. For example,Arg 73 and/or Arg 75 and/or Arg 76 can be substituted with a differentamino acid, preferably with Glu.

[0037] The “active variants” of LAG-3 are differentially splicedvariants as well as all primary gene transcripts, which derive fromalternative splicing mechanisms at different cleavage sites of the gene.Preferred variants are soluble or transmembrane proteins lacking the D3and/or D4 domains of the extracellular portion of LAG-3, optionallycontaining a few additional amino acids after the D2 or the D3 domain.

[0038] The expression of the transmembrane LAG-3 protein on the surfaceof a cell is verified by immunoreactive methods. The transmembraneprotein is recognized, for example, by the anti-LAG-3 antibodies 11E3(Deposit No. CNCM I-1612), 17B4 (Deposit No. CNCM I-1240) or 15A9(Deposit No. CNCM I-1239).

[0039] The present invention also refers to a mixture of polypeptidesand derivatives as said above.

[0040] When used in the present specification and claims, theexpressions “LAG-3”, “LAG-3 protein” or “LAG-3 molecule” are intended toinclude natural, synthetic and recombinant forms of the polypeptide aswell as all the definitions reported above.

[0041] The cells of the present invention can be selected from primaryor secondary cells. As used herein, the term primary cell includes cellspresent in a suspension of cells isolated from a vertebrate tissuesource (prior to their being plated i.e., attached to a tissue culturesubstrate such as a dish or flask), cells present in an explant derivedfrom tissue, both of the previous types of cells plated for the firsttime, and cell suspensions derived from these plated cells. The termsecondary cell or cell strain refers to cells at all subsequent steps inculturing. That is, the first time a plated primary cell is removed fromthe culture substrate and replated (passaged), it is referred to hereinas a secondary cell, as are all cells in subsequent passages. Secondarycells are cell strains which consist of secondary cells which have beenpassaged one or more times. A cell strain consists of secondary cellsthat: 1) have been passaged one or more times; 2) exhibit a finitenumber of mean population doublings in culture; 3) exhibit theproperties of contact-inhibited, anchorage dependent growth(anchorage-dependence does not apply to cells that are propagated insuspension culture); and 4) are not immortalized. A “clonal cell strain”is defined as a cell strain that is derived from a single founder cell.A “heterogenous cell strain” is defined as a cell strain that is derivedfrom two or more founder cells.

[0042] The present invention includes primary and secondary somaticcells, such as fibroblasts, keratinocytes, epithelial cells, endothelialcells, glial cells, neural cells, formed elements of the blood, musclecells, other somatic cells which can be cultured and somatic cellprecursors, which have been transfected with exogenous DNA which isstably integrated into their genomes or is expressed in the cellsepisomally. The resulting cells are referred to, respectively, astransfected primary cells and transfected secondary cells.

[0043] When the gene encoding a LAG-3 molecule is inserted intomammalian cells and the cells are transplanted into an allogeneic orxenogeneic host, they are recognized by the host immune system but animmune response is not mounted.

[0044] The host immune system becomes unable to reject the cells thatwould otherwise have been rejected had they not been engineered toexpress the LAG-3 molecule on their cell surface. LAG-3 can also beexpressed upon the cell surface of an unrelated cell type and mixed withthe cells or tissues to be transplanted with similar results to thosedescribed above. Thus, this invention relates to the transplantation ofcells, tissues, or organs without general immunosuppression.

[0045] These cells, tissues or organs are transplanted to provideproteins or perform certain functions to treat certain diseases. Theyare accepted by the host by the use of a technique in which LAG-3 ispresented to the host's immune system.

[0046] Prevention of graft rejection of specific cells, tissues ororgans can also be achieved in recipients by co-administration offibroblasts or other primary or secondary cells that have beenengineered to express LAG-3. This protective state may be due to localor general inhibition of mechanisms mediating immune responses. Thisprotective state may be due to anergy, deletion, non-responsiveness,tolerance or prevention of cell-mediated cytotoxicity. Once a protectivestate is established, it endures for extended periods of time, evenpermanently due to a phenomenon such as infectious tolerance (Qin etal., 1993).

[0047] The invention can be utilized for transplantation of cellstissues, organs or host cells to deliver genes or gene products for avariety of human medical needs.

[0048] LAG-3 gene expression can be induced by standard recombinant DNAtechniques or by techniques that employ homologous recombination toactivate the endogenous LAG-3 gene. The cell types can be obtained fromtransgenic animals that have the LAG-3 gene expressed in specifictissues or in unrelated cells by any method. Such cells could then bemixed with the cells in which protection from graft rejection isdesired. This suggests that local secretion of the immuno-protectivemolecule LAG-3 does not act systemically. LAG-3 transduced ,non-transformed fibroblasts yield similar responses in vivo. Theimmuno-protective effects of the innoculum of LAG-3 transducedfibroblasts are dose dependent but independent of the source of theLAG-3 molecule.

[0049] The co-administration of LAG-3 expressing cells inactivates donorT cells while at the same time preventing attack from the host immunesystem. This treatment induces specific anergy, tolerance or otherwiseprotection from cell-mediated cytotoxicity in the recipient which canthen result in a long-term change in the immune microenvironmentallowing protection against autoreactive T cells. This can be done by coadministration of a small number of allogeneic bone marrow cells from ahealthy donor along with human allogeneic cells that have beenengineered to express LAG-3. This results in a decrease in autoreactiveT cells through the development of microchimerism ( Delaney et al.,1996).

[0050] Humans with specific diseases or deficiencies can benefit fromthe allogeneic transplantation of many different cells, tissues ororgans. For example organs, such as liver, kidney, heart, pancreas,small bowel are commonly transplanted and cells such as islets, neuraltissue for the treatment of Parkinson's disease or focal epilepsy,hematopoietic stem cells as a treatment for chemotherapy or radiationtherapy, normal hepatocytes to treat hypercholesterolemia, cardiac cellsfor myocardial infarction, muscle cells for muscular dystrophy aresuitable for transplantation.

[0051] Allogeneic bone marrow transplants have been difficult toaccomplish for a variety of reasons. They include: graft rejection,infection due to opportunistic infections as a result of contaminationof the graft or immunosuppressive drugs, or other reasons. Rejection maybe due to the resistance of the recipient to bone marrow engraftment bydonors and the tendency of competent immune cells to attack therecipient i.e. (Graft-versus-host disease). GVHD may be controlled bythe depletion of the graft of T cells or the co-administration ofimmunosuppressant drugs. Engraftment is reduced when T cells areeliminated.

[0052] As a result of the T cell elimination, there is a higherincidence of graft failure. It is thought that they may provide animportant function for engraftment such as the elaboration of cytokines(Keman et al., 1987).

[0053] It has been shown that only small numbers of allogeneic buthealthy bone marrow cells can reduce or even prevent the occurrence ofautoimmune disease in experimental models. However, treatments such asthese result in graft-versus-host disease.

[0054] Bone marrow transplantation has also been used as a method toeradicate certain tumors. This is purportedly due to the ability ofallogeneic T cells to recognize and kill tumor tissue, e.g. ingraft-versus- leukemia.

[0055] In all the above cases general immunosuppression is avoided ifthe cells or organs to be transplanted are engineered with a geneencoding LAG-3, so as to express a LAG-3 protein when transplanted in ahost and induce graft protection.

[0056] A first problem in allogeneic transplantation is the lack oftransplantable human tissue and nowadays demand for organs far exceedssupply. Although still regarded as an experimental procedure,xenotransplantation is considered to be a viable alternative toallotransplantation. Animals such as pigs or baboons are now beingconsidered as organ or cell donors. Protection from graft rejection maybe essential for successful clinical use of organs from differentspecies. Host resistance can be overcome, at least partially, forexample, using antibodies against human CD4, CD8, NK cells, ormicroencapsulating the animal cells to be transplanted. According to thepresent invention, the animals can be transgenically altered to expressLAG-3 gene in certain cell types like the islet cells by the use of theinsulin gene promoter and targeting system or other tissue specificmaker system.

[0057] The expression of LAG-3 on tumor tissue is thought to play animportant role in the resistance to cell-mediated attack against tumortissue from the host's immune system.

[0058] According to a particular embodiment of the present invention,through the use of gene therapy or ex vivo treatment of a small amountof tumor tissue and reimplantation, the tumor tissue can be engineeredto express antisense LAG-3 molecules or ribozymes specific for LAG-3message. This would allow the immune system to react to the tissue andlearn to destroy it. A small amount of the tumor tissue can also betreated with an antibody to LAG-3 to prevent T cell inhibition inducedby LAG-3 and allow induction of cellular and humoral immunity againstit.

[0059] Gene therapy is now highly desirable for the treatment of avariety of diseases, including but not limited to adenosine deaminasedeficiency (ADA), sickle cell anemia, thalassemia, hemophilia, diabetes,alpha-antitripsin deficiency, brain disorders such as Alzheimer'sdisease and other illnesses such as growth disorders and heart diseases,for example those caused by alterations in the way cholesterol ismetabolized, and defects of the immune system.

[0060] Different cells can be used for transplantation in individuals inneed, for example myoblasts for dystrophin delivery, cells that secretematerial such as TPO, GH, EPO, Factor IX or other factors, blood cellsfor the treatment of inheritable blood disorders and other primary humanor animal cells, such as endothelial cells, epitelial cells, connectivetissue cells, fibroblasts, mesenchymal cells, mesothelial cells andparenchymal cells.

[0061] Results of gene therapy have not been very satisfying due to anumber of problems. Even the most advanced trials in which a young girlhas been treated with the gene for adenosine deaminase (ADA), thepatient still receives weekly injection of PEG-ADA for fear that thegene therapy alone is ineffective.

[0062] One of the shortcomings of gene therapy protocols today is therequirement of individual production of host cells to attempt to preventrejection of the cell by the host immune system. Gene therapy must beperformed on an individual by individual basis. Further, the expressionof transgenes is usually found to be transient due to the expression ofother viral proteins which engage the host immune system even throughthe use of autologous cells for gene therapy.

[0063] Crippled adenoviral vectors are used but these have problems dueto the other viral proteins that are expressed that evoke an immuneresponse. Large concentrations of virus, even a crippled one, stimulatean inflammatory response and an immune attack. The host cell immunesystem will remember the viral vector so that future administrationswill be even less effective.

[0064] Viral protein E1 deleted replication defective adenoviruses areroutinely employed in gene therapy protocols. Unfortunately, they haveonly transiently effective in adult, immunocompetent hosts presumably asa result of an immune response directed against adenoviral orrecombinant proteins (Kozarsky and Wilson, 1993; Barr et al., 1992;Stratford et al., 1992; Rosenfeld et al., 1992; Lemarchand et al.,1992). Therefore, there is a great need to develop new vectors that arenot so immunogenic or methods that allow for immunological protection ofthe genetically engineered cells.

[0065] These vectors are prepared at high titer up to 10¹¹ plaqueforming units per ml and infect many replicating and non-replicatingcells. Use replication defective adenoviruses to deliver physiologicallevels of recombinant protein to systemic circulation. The LAG-3 gene ispreferably under the transcriptional control of the ubiquitously activecellular EF1α promoter and the 4F2HC enhancer (Tripathy et al., 1994).

[0066] Attempts have been made to avoid this problem by encapsulatingthe cells and by immunosuppressing the host.

[0067] With the methods herein described, xenogeneic or allogeneic cellscan be used as gene therapy hosts expressing a LAG-3 molecule on theirsurface, so as to induce protection from graft rejection by the host'simmune system. They are, for example, myoblasts, fibroblasts,hematopoietic stem cells, embryonic stem cells, foetal liver cells,umbilical vein endothelial cells, or CHO cells. The gene therapy hostcells can also be engineered to express the herpes simplex thymidinekinase gene. Such cells can be specifically destroyed by addition ofgancylovir.

[0068] The tk-gancyclovir sensitive cells have a significant advantageover non-sensitive cells, in that they can be deleted at any time (Bi etal., 1993).

[0069] The universal host cell that is prepared according to the presentinvention to express LAG-3 and the Hsv-tk gene on its cell surface thusallows for the generation of a universal gene therapy host cell that canbe implanted without immunosuppression and can be destroyed at any pointshould its activity no longer be required.

[0070] The allogeneic or xenogeneic gene therapy host cells of thepresent invention can be engineered to express a transgene and/or theLAG-3 gene through the use any method of gene transfer such as but notlimited to replication defective viruses, adeno-associated virus, highefficiency retrovirus, direct injection of DNA into bone marrow,electroporation, calcium phosphate transfection, microinjection,encapsulation into liposomes or erythrocyte ghosts. Cells that expressLAG-3, such as myoblasts or CHO cells may be co-administered with cellsexpressing a protein of interest to be permanently engrafted. Iftransient LAG-3 exposure is adequate, then the LAG-3 transfectedmyoblasts or CHO cells can contain a suicide gene, such as tk, asreported above, so that they can be removed by treatment withgancyclovir.

[0071] This method can be used to restore normal function byadministration of a gene or gene product or the removal or inactivationof a gene that is dangerous to the body (such as an oncogene). This goalcan also be achieved by implanting cells that deliver ribozymes orantisense cDNA to inhibit the production of unwanted protein such as HIVproteins or growth factors. The method can be used to correct enzymedeficiencies such as Gaucher's disease or ADA deficiency.

[0072] According to a further embodiment the present invention isdirected to a method for gene therapy comprising: (i) inserting into thehuman or animal cells of choice the gene needed for therapy and the geneencoding a LAG-3 molecule; and (ii) introducing cells resulting fromstep (i) into the patient. Alternatively, the genes as above may beinserted into a tissue organ of a patient, in vivo, by directtransfection of the genes as such, or in a vehicle which targets suchtissue or organ.

[0073] For example, the claimed expression system allows for theinjection of a naked DNA or of a viral vector directly into a cell grouplike muscle cells or administered directly into the airways (forinstance to treat cystic fibrosis). The construct contains not only thegene of interest (such as the conductance regulator (CFTR cDNA) but alsothe LAG-3 gene to be co-expressed on the cell type to prevent thepossible humoral immune responses to, for example, the adenovirus capsidproteins which would limit the efficacy of repeat administrations. Genetransfer into hematopoietic stem cells can be used for theadministration of multi-drug resistance genes to combat one of the sideeffects of chemotherapy-suppression of rapidly dividing immune cells.Retroviral vectors can be used in combination with cytokines such asSteel factor, kit ligand, IL3, GM-CSF, IL6, G-CSF, LIF, IL12 toencourage stem cells to divide.

[0074] The cells of choice can be cotransfected with genes encodingother immune suppressive agents, such as IL10, TGFβ, Fas ligand, inaddition to the LAG-3 gene.

[0075] As a particular embodiment of the present invention, the methodsdescribed above are used to treat the recipient with a small number ofcells of interest engineered to express the LAG-3 gene which will makethe host tolerant to a next administration of cells, tissues or organsdue to infectious tolerance by the host's immune system.

[0076] The invention will now be described by way of illustration onlywith reference to the following examples:

EXAMPLE 1 Methods

[0077] Generation of CHO Cells Expressing Transmembrane LAG-3

[0078] LAG-3 cDNA was excised as a 1620 bp Xho fragment from pCDM8plasmid (Invitrogen San Diego Calif.) and purified by agarose gelelectrophoresis.

[0079] The fragment was subcloned into the pCLH3AXSV2DHFRhαIVSA (Dα)mammalian expression vector (FIG. 3) digested with Xho. CHO-DUKX (DHFR⁻)cells were transfected with the DαLAG-3 construct by CaPO₄ precipitationmethod. Transfected cells were grown in selection medium (MEM mediumwithout deoxy- and ribonucleotides +10% dialyzed fetal bovine serum +1%L-glutamine +0.02 μM metothrexate). The expression of LAG-3 was checkedby western blotting on lysed cell membrane preparations and periodicallyby flow cytometric analysis using anti-LAG-3 monoclonal antibody 17B4.

[0080] Transplantation of CHO Cells into Mice

[0081] Chinese hamster ovary (CHO) cells, either untransfected (wildtype) or transfected with full length human LAG-3 or human LH cDNA, weredetached from plastic flasks and suspended in Dulbecco's modification ofminimum essential medium (DMEM) at a concentration of 1.75×10⁷ cells/ml.Twenty-six C57BL/6 female mice aged 7-9 weeks were distributed into 7groups as indicated in Table 1 and 200 ml of the cell suspensionindicated, containing 3.5×10⁶ cells were injected subcutaneously in theright flank of each animal. In groups 3, 6 and 7 the same mouse receivedLAG-3-transfected cells in the right flank and controll cells(LH-transfected or untransfected) in the other. Four days after theinjection the mice were sacrificed by CO₂ inhalation and the skin wasopened to examine the site of injection. TABLE 1 Experimental groups No.of Mouse CHO cells injected CHO cells injected Group mice Identificationin the right flank in the left flank 1 5 1 to 5 wild type — 2 5  6 to 10LAG-3 — 3 5 11 to 15 LAG-3 wild type 4 2 16 and 17 LH — 5 5 18 to 22LAG-3 — 6 2 23 and 24 LAG-3 wild type 7 2 25 and 26 LAG-3 LH

[0082] Evaluation of Cytotoxicity Against CHO Cells

[0083] Five C57BL/6 female mice per group were injected s.c. with 4×10⁵CHO cells transfected either with human LH or human LAG-3 cDNA. After 14days the mice were sacrificed and the spleen were removed to obtainsplenocyte suspensions. Splenocyte suspensions (effectors) were dilutedin culture medium (RPMI1640+10% fetal bovine serum + antibiotics) at 10⁷cells/ml and plated in triplicate at different dilutions to obtain thevarious effector to target ratios; 2 plates were prepared for eachsuspension. Target cells at 5×10³ cells/100 μl (either LH- orLAG-3-trasnsfected cells), labelled with ⁵¹Cr, were added to the plates(one plate for each target). After 20 hours at 37° C., 20 μl ofsupernatant were taken from each well and the release of ⁵¹Cr wasevaluated by liquid scintillation. The cytotoxic activity was calculatedas percentage of lysis according to the following formula:$\% = {\frac{\left( {{CPMsample} - {CPMspont}} \right)}{\left( {{CPMmax} - {CPMspont}} \right)} \times 100}$

[0084] where spont and max indicate the wells with culture medium(spontaneous Cr release from the target cells) and Triton X 1% (maximumCr release) replacing the effector suspension, respectively.

Results

[0085] Transplantation of CHO Cells into Mice

[0086] Most mice receiving LAG-3-transfected cells showed a white noduleat the site of injection which did not appear in the animals treatedwith wild type CHO or in those receiving LH-transfected CHO cells. Theinjection of wild type CHO cells caused the appearence of a diffusehemorrhage in the injection site in the majority of the mice. Thisphenomenon was less evident in animals receiving LH-transfected cells.In the mice injected with both wild type and LAG-3 transfected CHO cellsat different sites this nodule was apparent only in the site injectedwith the latter while an hemorrhage appeared in the other site (Table2).

[0087] Histological analysis performed previously in a similarexperiment showed the presence of heterologous cells in the nodules. Theresults of the experiment are shown in the attached pictures (see Table1 for the identification of the mice) and summarized in Table 3. TABLE 2Frequency (number of positive findings/ Injected No. of total number offlanks injected): cells mice hemorrhage nodule LAG-3 10  1/10  9/10 wildtype 5 3/5 0/5 LH 2 1/2 1/2 LAG-3 + wild type* 7 0/7 (a) 5/7 (c) 7/7 (a)1/7 (c) LAG-3 + LH* 2 0/2 (b) 1/2 (a) 0/2 (b) 2/2 (a)

[0088] TABLE 3 Frequency (number of positive findings/ Injected totalnumber of flanks injected): cells hemorrhage nodule CHO wild type  8/12(67%)  0/12 (0%) CHO LH 1/4 (25%) 1/4 (25%) CHO LAG-3  2/19 (11%) 16/19(84%)

[0089] Cytotoxicity Against CHO Cells

[0090] The expression of LAG-3 on the surface of CHO cells did notaffect the ability of the mice to be immunologically primed against thexenogeneic cells. In fact, the splenocytes from mice injected withLAG-3-CHO cells lysed both target cells as efficiently as those frommice primed with LH-CHO cells and better than non-primed mice. Howeverthe expression of LAG-3 on the cell surface was associated with areduced sensitivity to the cytotoxic activity induced by immunization ofthe mice versus the target as can be seen by comparing the percentage oflysis in FIG. 1 and FIG. 2. The “natural” cytotoxicity, exerted bysplenocytes from non-primed mice, is not reduced by surface expressionof LAG-3. This indicates that LAG-3 surface expression reduces theefferent branch of cytotoxic T lymphocyte (CTL) activity. CTL are one ofthe effectors playing a main role in the rejection of transplantatedorgans (G. Berke, 1993) thus the inhibition of their function canprolong the survival of allografts.

REFERENCES

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[0097] 7. Susuki et al., J. Exp. Med., 182: 477-486, 1995

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[0100] 10. Subraimani et al., Mol. Cell. Biol., 1: 854-864, 1981

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[0104] 14. Qin et al., Science, 259: 974, 1993

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[0106] 16. Kernan et al., Transplantation, 43: 842, 1987

[0107] 17. Kozarsky and Wilson, Current Opinions Genet. Dev., 3: 499,1993

[0108] 18. Barr et al., Gene Therapy, 1: 51, 1992

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1. Genetically engineered cell comprising DNA encoding a transmembraneLAG-3 protein on its surface resulting in the protection from graftrejection by a host's immune system.
 2. A cell as claimed in claim 1,wherein the DNA encoding a LAG-3 protein is exogenous.
 3. A cell asclaimed in claim 1, wherein the DNA encoding a LAG-3 protein isendogenous and its expression is activated or modified through thetargeted insertion of a regulatory sequence and/or an amplifiable geneby homologous recombination.
 4. A cell as claimed in any of claims 1 to3 which is part of a tissue or organ to be transplanted.
 5. A cell asclaimed in any of claims 1 to 3, which is a gene therapy host cell.
 6. Acell as claimed in claim 5, wherein gene therapy is somatic or “ex vivo”gene therapy.
 7. A cell as claimed in any of claims 1 to 6, furthercomprising an additional immuno-suppressive agent, such as IL-10, TGFβor Fas ligand.
 8. A cell as claimed in any of claims 1 to 7, furthercomprising the thymidine Kinase (tk) gene to be sensitive to thetk-gancyclovir suicide system.
 9. A cell as claimed in any of claims 1to 8, deriving from transgenic animals.
 10. A cell as claimed in any ofclaims 1 to 9, selected from myoblasts, fibroblasts, hematopoietic stemcells, embryonic stem cells, foetal liver cells, umbilical veinendothelial cells or CHO cells.
 11. A cell as claimed in any of claims 5to 10, further comprising exogenous DNA encoding a therapeutic agent ofinterest.
 12. A cell as claimed in any of claims 5 or 10, furthercomprising exogenous DNA encoding a regulatory sequence and or anamplifiable gene for activating or modifying the expression of anendogenous gene of interest.
 13. Use of a transmembrane LAG-3 proteinexpressed on the surface of a cell in the manufacture of a medicament toinduce protection from graft rejection by a host's immune system. 14.Use of a cell as described in any of claims 1 to 12 in the manufactureof a medicament to induce protection from graft rejection by a host'simmune system.
 15. Use of a cell as described in any of claims 1 to 10in the manufacture of a medicament to be mixed with cells, tissues ororgans to be transplanted, to induce protection from graft rejection bya host's immune system.
 16. Process for inducing specific protectionfrom graft rejection of transplanted cells, tissues or organs by ahost's immune system, comprising treatment of the host patient with acell according to any of claims 1 to
 12. 17. Process as claimed in claim16, wherein the cell is part of a tissue or organ to be transplanted.18. Process as claimed in claim 16, wherein the cell is a gene therapyhost cell.
 19. Process as claimed in claim 18, comprising: (i) insertinginto the host cell of choice the gene needed for therapy and the geneencoding the LAG-3 molecule and (ii) introducing cells resulting fromstep (i) into a host patient in need.
 20. Process as claimed in any ofclaims 16 to 19, comprising the treatment of a host patient in need witha small number of cells engineered to express the LAG-3 gene, to induceprotection from graft rejection by the host's immune system, to a nextadministration of the cells, tissues or organs to be transplanted. 21.Process for allowing rejection of tumor tissue, comprising engineeringsaid tumor tissue cells to express antisense LAG-3 molecules orribozymes specific for LAG-3 message.
 22. Genetically engineered cellcomprising DNA encoding a transmembrane LAG-3 protein on its surface foruse as a medicament.